Blood testing device

ABSTRACT

This invention relates to the measurement of various haemostatis, clotting, and the like properties of blood.

This is a division of application Ser. No. 08/743,911, filed Nov. 4,1996, now U.S. Pat. No. 5,925,569, which was a continuation ofapplication Ser. No. 07/314,663, filed Feb. 3, 1989, now U.S. Pat. No.5,665,311, which was a US national phase of PCT/GB87/00633, filed Sep.10, 1987. Priority is claimed in this case from each of the above-listedapplications.

This invention relates to the measurement of various haemostatis,clotting., and the like properties of blood, and specifically toapparatus enabling measurements of such quantities to be made on blood.A device of this kind is disclosed in European Patent Specification No.129425.

In that specification there is disclosed apparatus for the investigationof blood, comprising a columnar reservoir for holding the blood, anelongate tube leading from the reservoir to a waste blood holdingvessel, and means for introducing into the reservoir a fluid immisciblewith blood to displace the blood through the tube, while simultaneouslystirring the blood in the columnar reservoir (hereinafter referred to as"apparatus of the type described").

I have now discovered a number of modifications and improvements to theapparatus disclosed in European Patent Specification No. 129425, whichnot only enable more reliable and more reproducible measurements to bemade of clotting and haemostatis, but also enable a number of otherquantities to be measured.

In accordance with a first aspect of the invention, apparatus of thetype described includes a pair of parallel channels, each channelcomprising a columnar reservoir, elongate tube, waste blood holdingvessel, and means for introducing a displacing fluid, as describedabove.

The use of two parallel channels enables a number of measurements to bemade which are not possible with a simple single channel instrument. Forexample, in a preferred embodiment of this aspect of the invention, eachchannel has a punching station, at which, as disclosed in EuropeanPatent Specification No. 129425, a through-hole is punched through theelongate tube, to simulate bleeding. So that the size of the holes inthe two tubes is exactly the same, both holes may be punched by a singleparallel-sided punching needle. In an alternative embodiment, twoexactly similar needles may be provided at corresponding places in thetubes of the two channels.

The use of parallel channels is particularly advantageous, because itenables measurements of clotting and haemostasis kinds to be carried outon whole blood. Thus, in one embodiment, the columnar reservoir of bothchannels is filled with native blood, and caused to flow through therespective elongate tube, by displacement. Both channels are pierced,and the time take for haemostatis to occur (i.e. the formation of aplatelet plug in the two holes) is measured in both channels. Becausethe test is carried out in both channels using whole blood, the plateletplug formed simulates very closely platelet plugs formed in arterialbleeding.

An anticoagulant agent is then introduced in to the blood flowing in oneof the channels. This prevents the blood flowing in that channel fromclotting, so that thrombolysis of the platelet plug in that channel canbe investigated but does not interfere with the haemostatic plugformation because the haemostatic plug has already been formed when theanticoagulant is introduced. A preferred anticoagulant is heparin,because of its low interference with the thrombolytic process.

After a period of time the blood in the channel which is notanticoagulated will clot, and this can be measured by appropriatepressure transducers. In the channel to which the anticoagulant has beenadded, the platelet plug will eventually dissolve, and thereforethrombolysis time can be measured.

The two-channel instrument of the first embodiment of the presentinvention is also useful in other clinical diagnostic techniques, forexample to determine the effects of various pharmaceuticals substanceson clotting, haemostatis and thrombolysis. Native blood containingsaline (control) may be utilised in one channel of the apparatus, andidentical blood containing the pharmaceutical substance to beinvestigated in the other, and thus the effect on the various propertiesof the blood can be monitored under closely controlled conditions.

In a second aspect, the invention is concerned with the effect ofvarious substances, for example collagen, on haemostasis/thrombosis. Forexample, a small piece of collagen, such as a piece of a surgicalsuture, may be placed within the elongate tube leading from the columnarreservoir to the blood holding vessel of apparatus of the typedescribed, and the blood caused to flow over the substance.

The mechanism of platelet thrombus formation on the surface ofsubstances such as collagen is similar to that which occurs during thenormal passage of blood through injured blood vessels in the body.Collagen fibres are exposed when blood vessels are ruptured and it is ofconsiderable clinical interest to investigate in detail the effect ofcollagen during thrombus formation. In particular, it has beenestablished by electron micrography that thrombus formation takes placeby means of the build up of a monolayer of platelets on the collagensurface, followed by subsequent platelet adhesion to succeeding layers.Electron micrography is an expensive and time consuming technique, andin a second aspect of the invention, a method and apparatus are providedfor enabling the investigation of platelets on substances such ascollagen or other bio-compatible materials to be carried out, usingrelatively simply apparatus.

In accordance with this aspect of the invention, a piece of thesubstance such as collagen is provided in the elongate tube of theapparatus, and the progress of thrombus formation is followed byobserving blood flow in the tube, for example by the measurement ofpressure, as disclosed hereinafter, as the formation of a thrombus onthe substance causes a characteristic pattern of occlusion of the tube.

In accordance with a third aspect of the invention, three parallelchannels may be provided, to enable the effect of substances such ascollagen on thrombus formation to be determined, whilst othermeasurements are also being performed, for example haemostatis and bloodclotting measurements.

A fourth aspect of the invention relates to the method by which bloodflow in apparatus of the type described is measured and controlled. InEuropean Patent Application No. 129425, a pressure transducer isutilised, to measure variation of pressure in the blood, as the holesare punched, and subsequently occluded by platelet plugs. The pressuretransducer however is positioned upstream of the punching station, sothat the only measurement possible with regarding to clotting in thetube is the point at which blood flow in the tubes ceases completely. Inaccordance with the fourth aspect of the invention, apparatus of thetype described includes a pressure transducer downstream of the elongatetube, and preferably disposed so as to measure pressure in the wasteblood holding vessel, whereby the process of blood clotting can befollowed. Thus, as the blood clot in the tube grows in size, flowthrough the tube diminishes steadily, and thus the pressure in the wasteblood reservoir falls. As in European Patent Application No. 129425, thewaste holding vessel is; in this embodiment, filled with a fluidimmiscible with the blood, which is displaced by the entry of the blood.

A fifth aspect of the invention is concerned with the mechanism forcausing the blood to pass through the elongate tube. In European PatentApplication No. 129425, the blood is displaced by a fluid immiscibletherewith, for example paraffin oil, which is supplied to the columnarcontainer using a syringe pump. This arrangement is somewhatdisadvantageous in practice, because it tends to result in a pressureprofile which varies unacceptably, and is also rather difficult tocontrol.

In accordance with a sixth aspect of the invention, apparatus of thetype described includes means for introducing the immiscible fluid intothe columnar container, comprising a pressurisable vessel for theimmiscible fluid, means for applying a constant pressure head to thepressurisable container, and a capillary tube leading from thepressurisable vessel to the columnar container, the capillary tubehaving a resistance to flow substantially greater than that of the tubeleading from the columnar container to the waste blood holding vessel.Because in this arrangement, the greater part of the pressure dropacross the apparatus takes place across the capillary tube, rather thanthe tube which is pierced, a constant pressure applied to the pressurevessel results in a substantially contant flow of the displacing fluidinto the columnar container. Thus, the flow of blood through theapparatus can be readily controlled, by controlling the pressure in thepressurisable container. This may be applied using, for example, atwin-head pump, or a gas cylinder.

A seventh aspect of the invention relates to the nature of the columnarcontainer itself. In the apparatus disclosed in European PatentSpecification No. 129425, the columnar container is essentially a tubeprovided with a fluid inlet and a fluid outlet at its bottom part, for,respectively, introduction of the displacing fluid, and the outflow ofblood. One disadvantage of this arrangement is that it necessitatestransfer of the blood from one container to another, after it has beentaken from the patient, and this can result in the initiation of theclotting process. Furthermore, the tube arrangement includes a number ofsharp corners over which the blood must flow, and these and theturbulence caused also can initiate the clotting/haemostasis process. Inthe seventh aspect of the invention, the columnar container ispreferably a plastics syringe, which can be used to take a blood samplefrom a patient in the ordinary way, and from which the hypodermic needlecan then be removed, so that the elongate tube leading to the wasteblood holding vessel can be attached. The apparatus preferably includesa mounting block for the syringe, which may include temperature controlmeans, for example a water jacket, or a thermostatically controlledheating element. In devices according to the invention having two orthree parallel channels, the mounting block will generally be adaptedfor mounting a syringe for each channel.

The support block may also preferably comprise a port or septum, so thata hypodermic needle may be introduced into the syringe, through itswall, for introduction of the displacement fluid. The displacement fluidwill normally be less dense than blood, and accordingly the port ispreferably positioned close to the bottom of the hypodermic syringe, sothat introduction of the displacement fluid causes effective stirring ofthe blood. The elongate tube leading from the syringe to the waste bloodvessel is preferably attached by a LUER-type fitting, and by this methodvirtually all parts of the apparatus which come into contact with bloodare rendered disposable. Thus, the syringe, tubing and waste bloodholding vessel may all be discarded after use.

Yet a further aspect of the invention is concerned with theinvestigation of thrombolysis. As indicated above, by carrying out thesecond phase of the measurements on anticoagulated blood, it is possibleto follow the progress of thrombolysis in blood, and to investigate theeffect of various agents on thrombolysis. Although haemostatis andclotting are relatively quick to occur, thrombolysis will generallyrequire a much longer time for completion, for example from fifteen toforty minutes. If the blood is caused to flow in the elongate tube forthe whole of this period, substantial quantities of blood are required.However, once the anticoagulant has been added, it is no longernecessary for the flow of blood to be maintained in the tube, providedthat the pressure of the blood remains constant.

Accordingly, in this further aspect of the invention, there is provideda valve, for example a needle valve, for closing the outlet of the wasteblood holding vessel, so that after anticoagulated blood has passedthrough the whole of the elongate tube, the valve can be closed, suchthat pressure in the system can be maintained, without the loss of bloodtherefrom. The progress of thrombolysis can then be measured, as above,using a pressure transducer in the waste blood holding vessel. Thisarrangement is particularly advantageously used with the arrangementdescribed above of a pressurised vessel for driving the displacementfluid in the columnar blood container. In this preferred embodiment,means may be provided for reducing the pressure applied to thepressurisable vessel, when the valve is closed. Since the internalpressure in the blood-containing parts of the system (the columnarcontainer, elongate tube and waste blood holding vessel) issubstantially below that in the pressurisable container, closing of thevalve would result in the overall rise in pressure in the system,because of equalisation of the pressure in the blood-containing parts,and the displacement fluid containing parts. In this preferredembodiment of the invention therefore, means are preferably provided forapplying a reduced pressure to the displacement fluid, after closure ofthe valve. In one embodiment, the means for applying the reducedpressure may take the form of two pressurisable containers, pressurisedto different pressures, and alternatively switchable to provide thepressure to drive the displacement fluid.

Although all of the various aspects of the invention are preferablyembodied in the same apparatus, it should be understood that each aspectmay be utilised independently, or the various aspects may be utilised inany desired combination.

A particularly preferred embodiment of apparatus incorporating all ofthe above aspects of the invention will now be described, with referenceto the accompanying drawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing one channel of a two-channel bloodanalysis device,

FIG. 2 is a schematic diagram of the punching station of a two-channeldevice,

FIG. 3 shows a preferred pressurised vessel arrangement,

FIG. 4 shows samples of haemostatis, clotting, and thrombolysis tracesobtained with the instrument of FIGS. 1 and 2, and

FIGS. 5A and 5B illustrate the investigation of platelet, collageninteraction.

Referring first to FIG. 1, one channel of a two-channel instrumentcomprises a columnar container for holding blood, in the form of a 2 mlhypodermic syringe 1, made of a disposable plastics material. A elongatepolyethylene tube, having an internal. diameter of 0.5 mm, and a lengthof approximately 30 cm is connected to the syringe 1 by means ofLUER-type connector 3. The tube 2 is inserted through a pressure-tightrubber seal 4 in a waste blood holding vessel 5. At the commencement ofany particular investigation, the waste blood holding vessel 5 is filledwith paraffin oil 6, and the level of blood 7 in the vessel rises duringthe investigation.

The syringe 1 is received in a syringe mounting block 8, provided withan electrical heating element and thermostat (not shown) to maintain theheating block 8 at a constant temperature of 37° C. The plunger 9 of thesyringe 1 is secured by a lock (not shown), to prevent its backwardmovement, on pressurisation of the blood in the syringe.

The polyethylene tube 2 is threaded through a punching station,generally of the kind described in European Patent Specifiction No.129425, except for the modification that support is provided for twosuch polyethylene tubes, and a punching needle 10 is of a sufficientlength, and has sufficient movement, to punch through both sides of thetubes 2 of two channels. This is illustrated more clearly in FIG. 2.Needle 10 is attached to a plunger 11, which may be manually actuated,but is preferably actuated by means of a solenoid, or hydraulic orpneumatic actuator. The shank of the punching needle 10 has a diameterof 0.15 mm.

Means (not shown) are provided for enabling the supply of warm salinesolution to the punching station, surrounding the tubes, to wash awaythe blood, as simulated bleeding occurs from holes 12 when the tube ispunched. An outlet is provided for the saline solution, and means areprovided associated with outlet for causing the saline solution to flowbetween a light-emitting diode, emitting green light, and a siliconphotodiode.

The waste blood holding vessel 5 has three parts, a base 5a, a centraltubular part 5b and an upper part 5c. The upper 5c has an opening 19 towhich is affixed a pressure transducer 20. An outlet 21 for paraffin oilis also provided in the upper part 5c. A valve 22 is provided forclosing outlet 21 from the vessel 5.

Parts 5a, 5b and 5c of the vessel 5 are assembled as a push-fit, and aresealed by "O" rings 24 and 25.

FIG. 3 illustrates schematically an arrangement for supplying animmiscible fluid (paraffin oil) under pressure to the two channels ofthe instrument.

The apparatus for FIG. 3 includes two pressurisable vessels 30 and 31,and a three-way tap 32. The three-way tap 32 is connected to a lowpressure gas supply line 33, and a high pressure gas supply line 34. Inthe embodiment shown, the pressure in line 33 is arranged to be 60 torr,and that in line 34, 360 torr (all pressures referred to herein aregauge pressures) regulators 35 and 36 respectively are provided tomaintain the pressure in lines 33 and 34 of the desired level. Thethree-way valve 32 can be turned between a first position, as shown, inwhich pressure line 33 is connected directly to pressure vessel 30, vialine 38, and pressure line 34 is connected directly to vessel 31, vialine 39, and a second position, in which pressure line 34 is connectedto both pressure vessels 30 and 31.

Each of vessel 30 and 31 is filled with paraffin oil, and is providedwith a respective capillary tube 40, 41 having an internal diameter ofapproximately 0.18 mm, and a length of approximately 30 cm, tohypodermic needles 42 and 43 respectively.

Means 46 are provided for applying gas under pressure to both pressureregulators 35 and 36. The means 46 may be a pump, or a cylinder ofpressurised gas.

In operation of the apparatus, two blood samples are taken from thepatient using respective syringes 1 of the two-channels of theapparatus. The syringes 1 are placed in the heating block 8 of theapparatus, and the luer-lock connector 3 is connected to the syringeoutlet. The respective tubes 2 are then passed through the piercingstation, and through rubber seals 4 of two waste blood holding vessels5.

Each of the respective syringes 1 is then-pierced through its side witha respective hypodermic needle 42, 43, connected to paraffin oilsupplies in pressurised vessels 30 and 31 respectively. A pressure of360 torr is applied to both pressure vessels 30 and 31, which causesparaffin oil to be displaced into the syringes 1 at a rate ofapproximately 0.1 m per minute. The inflowing paraffin oil preventsedimentation of red cells in the blood, and displaces the blood throughthe tubing 2. The pressure at the pressure transducer 20 is observed,until it is steady, with a steady flow of blood entering vessel 5. Thetubes 2 of both channels are then simultaneously pierced with the singleneedle 10, and the pressure at transducer 20 is observed further. Aconfirmatory check of bleeding through holes 12 is carried out notingthe signal received at the silicon photodiode. Bleeding from holes 12causes the signal to be much reduced, so that on-set and cessation ofbleeding can be noted and confirmed.

FIG. 4 illustrates a typical pressure trace obtained for channels I andII of two-channel apparatus.

In FIG. 4, the trace shown for channel II illustrates the normal processof haemostatis and clotting, when no anticoagulant is used. Point A isFIG. 4 represents the point at which the tubes 2 are pierced, and as canbe seen, the pressure in vessel 5 descreased rapidly, and then slowlyrises over a period of approximately two minutes, as haemostatic plugsare formed in holes 12. The pressure in channel II then rises again toits original level of 60 torr, until clotting occurs in tube 2. Theon-set of clotting can be seen at point B, and clotting is complete atpoint C.

In channel I of the apparatus, the progress of haemostatis is identicalwith that in channel II, but after the haemostatic plugs have beenformed, at point D in FIG. 4, an anticoagulant is introduced into theblood in columnar container 1. In practice, this is carried out byintroducing into the paraffin oil flowing into the syringe 1 an amountof heparin such as to give a final concentration of 5 U/ml in the blood.Following injection of the anticoagulant, the blood in channel I doesnot clot, but continues to flow in the tube. To minimize wastage ofblood therefore, after sufficient time has been allowed forheparin-containing blood to pass through the system, the valve 22 isclosed, and valve 32 is operated, so as to reduce the pressure appliedto pressure vessel 30 to 60 torr.

By this method, the progress of the breakdown of, platelet plugs can beobserved, and the effect of various drugs on thrombolysis can also benoted. The breakdown of the haemostatic plugs is shown not only bypressure drop at point E in FIG. 4, but also by the silicon photodiodereferred to above. The on-set of bleeding, as sensed by the photodiode,at point A is used to start a electronic clock, when blood appearing inthe light pathway reaches a pre-set sensitivity threshold, and decreasesthe photodiode output below that threshold stops the Quartz clock, toshow the elapsed (thrombolysis) time.

In the embodiment illustrated in FIG. 5, the arrangement is generallythe same as illustrated in FIG. 1, except that it is not necessary toutilise the needle 10 to punch the tube 2.

As illustrated in FIGS. 5A and 5B illustrates, a short piece of collagenfiber (surgical catgut), having a diameter of approximately 0.15 mm isplaced within the tube 2, before the blood supply is started. Plateletsadhere and aggregate on the surface of the catgut, as illustrated inFIG. 5A, and eventually occlude the tube. The rate and extent of thedecline of the pressure in the vessel 5 indicate the platelet adhesionto the collagen, and subsequent thrombus formation.

This procedure allows the quantative measurement of platelet functionwhich is most characteristically changed in certain disorders (forexample Von Willebrand disease, and by drugs, for example aspirin).

In accordance with the invention, apparatus for the determination of theeffects of collagen and the like on thrombus formation may omit thepunching means 12. It is preferred however that apparatus in accordancewith the invention comprises three substantially identical channels, allas illustrated in FIG. 1. The tubes 2 of two of the channels passthrough the punching station, but the other does not, and is intended toreceive the piece of collagen fiber.

The invention also provides methods for the determination of clotting,haemostatis and thrombolysis factors of blood, using apparatus asdescribed above.

It is a particular advantage of the apparatus described above that invivo bleeding is simulated very closely, as well as the subsequentrestorative processes, i.e. haemostatis, blood clotting, and clotdisolution (thrombolysis). The device disclosed enables the simultaneousmeasurement of these three factors in a simple, reproducable way, underconditions close to those of haemodynamic stress, and in which a largeamount of the blood utilised is saved. Furthermore, non-anticoagulated(native) blood can be utilised for all of these measurements, and all ofthe various measurements can be made from a blood sample of no more than5 ml. Furthermore, evaluation of drugs which modify any of the threeabove processes can be carried out in a setting that is much morephysiologically relevant than other in vitro methods used previously inhaemostatis research, such as platelet aggregometry.

We claim:
 1. A method for the investigation of blood,comprising:providing a columnar reservoir for holding blood to beinvestigated, providing an elongate conduit leading from the reservoirto a waste blood holding vessel, flowing blood through said conduit,including introducing into the reservoir a fluid immiscible with bloodto displace the blood through the conduit, while simultaneouslyagitating the blood in the columnar reservoir, initiating the process ofhaemostasis within said conduit with means for initiating haemostasisthat is one of (1) operatively coupled to said conduit and (2) disposedin said conduit, after a haemostatic plug has formed in said conduit,introducing an anticoagulant into the blood upstream of the haemostaticplug, closing an outlet of the waste blood holding vessel, so as tomaintain pressure in the system without a loss of blood, reducing apressure applied to the immiscible fluid, and monitoring the breakdownof the haemostatic plug by one of monitoring pressure and sensing bloodflow downstream of said haemostatic plug.
 2. A method as in claim 1further comprising introducing a substance, a thrombolytic effect ofwhich is to be investigated, into the blood upstream of the haemostaticplug.
 3. A method as claimed in claim 1, wherein the step of reducing apressure applied to the immiscible fluid comprises switching between twopressurizable containers pressurized to different pressures, to drivethe displacement fluid.
 4. A method as claimed in claim 1, furthercomprising the steps of:providing a second columnar reservoir forholding blood, providing a second elongate conduit leading from thesecond reservoir to the waste blood holding vessel, flowing bloodthrough said second conduit, including introducing into the secondreservoir a fluid immiscible with blood to displace the blood throughthe second conduit, while simultaneously agitating the blood in thesecond reservoir, initiating the process of haemostasis within saidsecond conduit with means for initiating haemostasis that is one of (1)operatively coupled to said second conduit and (2) disposed in saidsecond conduit, and one of monitoring pressure and sensing blood flowdownstream of said haemostatic plug in said second conduit, thereby todefine a control for comparison with the monitored haemostatic plugbreakdown.
 5. Apparatus for the investigation of blood, comprising acolumnar reservoir for holding the blood, an elongate tube leading fromthe reservoir to a waste blood holding vessel, means for introducinginto the reservoir a fluid immiscible with blood to displace the bloodthrough the tube, while simultaneously stirring the blood in thecolumnar reservoir, a valve for closing an outlet of the waste bloodholding vessel, so that after anticoagulated blood has passed throughthe whole of the elongate tube, the valve can be closed, maintaining thepressure in the system without a loss of blood,means for applying thereduced pressure to the displacement fluid, after closure of the valve,wherein the means for applying the reduced pressure comprises twopressurizable containers, means for pressurizing the containers todifferent pressures, and means for alternatively switching the pressurefrom the two containers to drive the displacement fluid.
 6. An apparatusfor measuring physical properties of blood comprising:at least tworeservoirs for holding blood; at least two elongate tubes connected atone end to a respective reservoir; a punching station located along saidtwo tubes which permits simultaneous measurement of blood properties insaid elongated tubes; waste receptacle means connected to remaining endsof said elongated tubes for collecting blood flowing through saidelongated tubes; and, means for introducing a displacing medium intosaid reservoirs for stirring the contents of said reservoirs and forcingsaid fluid through said elongated tubes past said punching stationpermitting a comparison between physical characteristics of bloodpassing through said punching station, wherein said means forintroducing said displacing medium into said reservoirs comprises: apressurizable vessel for holding said immiscible fluid; means forapplying a constant pressure to said pressurizable vessel; and, acapillary tube leading from said pressurizable vessel to saidreservoirs, said capillary tube having a resistance to flowsubstantially greater than that of said elongate tubes, whereby asubstantially constant flow through said elongate tubes occurs.
 7. Anapparatus for measuring physical properties of blood comprising:at leasttwo reservoirs for holding blood; at least two elongate tubes connectedat one end to a respective reservoir; a punching station located alongsaid two tubes which permits simultaneous measurement of bloodproperties in said elongated tubes; waste receptacle means connected toremaining ends of said elongated tubes for collecting blood flowingthrough said elongated tubes; means for introducing a displacing mediuminto said reservoirs for stirring the contents of said reservoirs andforcing said blood through said elongated tubes past said punchingstation permitting a comparison between physical characteristics ofblood passing through said punching station, a three-way tap connectedto a source of high pressure and a source of low pressure through firstand second regulators; first and second pressurizable vessels, saidfirst vessel having a first inlet connected to said three-way tap, saidsecond vessel having an inlet connected to said source of high pressure;and, first and second capillary tubes connecting said first and secondvessels to said reservoirs.
 8. Apparatus for the investigation of blood,comprising a columnar reservoir for holding the blood, an elongate tubeleading from the reservoir to a waste blood holding vessel, means forintroducing into the reservoir a fluid immiscible with blood to displacethe blood through the tube, while simultaneously stirring the blood inthe columnar reservoir, a valve for closing an outlet of the waste bloodholding vessel, so that after anticoagulated blood has passed throughthe whole of the elongate tube, the valve can be closed, maintaining thepressure in the system without a loss of blood,wherein the means forintroducing the immiscible fluid into the columnar reservoir comprises apressurizable vessel for the immiscible fluid, means for applying aconstant pressure head to said pressurizable vessel, and means forreducing the pressure applied to the pressurizable vessel when the valveis closed.